Use of the glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol as a probe to investigate hypertrehalosemic hormone-mediated change in carbohydrate and lipid metabolism in the cockroach, Periplaneta americana

The glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DIA) was used to investigate the action of hypertrehalosemic hormone (HTH) on carbohydrate, neutral lipid, and phospholipid metabolism in the hemolymph and fat body of the cockroach, Periplaneta americana. DIA blocked the hypertrehalosemic and hyperglycemic effects of HTH, as well as the decrease in hemolymph neutral lipid and phospholipid normally induced by HTH. DIA diminished the accumulation of neutral lipid in the fat body formed under the influence of HTH and partly blocked the decrease in fat body phospholipid evoked by HTH. The increased incorporation of (14)C-glucose into fat body triacylglycerol in the presence of HTH was decreased by more than two-thirds when DIA was coinjected with the hormone. The results suggest that glucose derived from hemolymph trehalose is an important contributor to the formation of the glycerol backbone of newly formed triacylglycerol in the fat body.     

Evidence for the participation of arachidonic acid metabolites in trehalose efflux from the hormone activated fat body of the cockroach (Periplaneta americana)

The hypertrehalosemic hormones, HTH-I and HTH-II, activate trehalose synthesis and increase the rate of sugar efflux from Periplaneta americana fat body in vitro. These processes are unaffected by the diacylglycerol, 1-oleyl-2-acetyl-sn-glycerol, an activator of protein kinase C. Similarly, H-7 and spingosine, inhibitors of protein kinase C, are also inactive against trehalose efflux. The possibility that diacylglycerol lipase might generate an active fatty acid species was ruled out because of the failure of the inhibitor RHC-80267 to inhibit trehalose efflux. Activation of trehalose efflux from the intact fat body by HTH-I was strongly inhibited in a concentration dependent manner by the cyclooxygenase inhibitors indomethacin and diclofenac, but not by acetylsalicylic acid. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, also blocked HTH-I activated trehalose efflux in a concentration dependent fashion. The phospholipase A(2) inhibitors mepacrine and 4'-bromophenacyl bromide were also effective in decreasing the efflux of trehalose from HTH-I challenged fat body. The data suggest possible roles for arachidonic acid metabolites in the regulation of trehalose synthesis and in the efflux of the sugar from the fat body.     

Carbohydrate metabolism during starvation in the silkworm Bombyx mori

The effect of starvation on carbohydrate metabolism in the last instar larvae of the silkworm Bombyx mori was examined. Trehalose concentration in the hemolymph increased slightly during the first 6 h of starvation and decreased thereafter, whereas glucose concentration decreased rapidly immediately after diet deprivation. Starvation-induced hypertrehalosemia was completely inhibited by neck ligation, suggesting that starvation stimulates the release of a hypertrehalosemic factor(s) from the head. The percentage of active glycogen phosphorylase in the fat body increased within 3 h of starvation and its glycogen content decreased gradually. These observations suggest that production of trehalose from glycogen is enhanced in starved larvae. However, hypertrehalosemia during starvation cannot be explained by the increased supply of trehalose into hemolymph alone, as similar changes in phosphorylase activity and glycogen content in the fat body were observed in neck-ligated larvae, in which hemolymph trehalose concentration did not increase but decreased gradually. When injected into larvae, trehalose disappeared from hemolymph at a rate about 40% lower in starved larvae than neck-ligated larvae. The hemolymph lipid concentration increased during starvation, suggesting that an increased supply of lipids to tissues suppresses the consumption of hemolymph trehalose and this is an important factor in hypertrehalosemia.     

Uptake of cholesterol and cholestanol by the intestine,hemolymph, and fat body of Heliothis zea

The effect of the concentration and structure of dietary sterol on its uptake and distribution in the intestine, hemolymph and fat body was studied in sixth-instar larvae of Heliothis zea. When cholesterol (cholest-5-en-3β-ol) was inoculated per os into the foregut of larvae, it was rapidly taken up by the intestine. Some of the dietary sterol then passed into the hemolymph, primarily via the midgut, during at least the first 9 h after inoculation, while at least 7% of the dose remained associated with the intestine. The amount of dietary sterol per 0.10 g of hemolymph increased until it reached 3–6% of the dose after 9 h. The amount of sterol per 0.10 g of the fat body increased to as much as 5% of the dose after 10 h. As the concentration of sterol in the dose increased from 0.3 to 15 μg/4 μl, the amount of sterol associated with the intestine, hemolymph, and fat body also increased. When cholesterol was inoculated intrahemocoelically, instead of per os, the amount of sterol in the hemolymph decreased, for at least the first 8 h after inoculation, and may have been absorbed, at least in part, by the intestine. The absence of a double bond in cholestanol (5α-cholestan-3β-ol) had no significant effect, at least 5 h after inoculation, on the uptake and distribution of this sterol in the intestine, hemolymph, and fat body of the larva. The results of this study indicate that although larvae of H. zea fed cholestanol have a slower rate of growth than those fed cholesterol, this may be due to differences in the utilization of the two sterols rather than to differences in their uptake by the tissues.     

Membrane-bound sorbitol 6-phosphatase in fat body cells controls the dynamics of sorbitol 6-phosphate, a major hemolymph sugar in the silkworm

Sorbitol 6-phosphate (S6P) is one of two major sugars (another is trehalose) in the larval hemolymph of Bombyx mori, and its amount dramatically decreases concomitantly with the onset of prepupal period. In the last (fifth) instar larvae, the amount of S6P is approximately 30 micromol/larva at its maximum and decreases to less than 1 micromol at the wandering stage. Incubation of fat bodies of wandering larvae with S6P generates sorbitol in the medium, while S6P in the medium decreases, indicating that fat body possesses sorbitol 6-phosphatase (S6Pase) activity. S6Pase activity in the fat body remains low during the feeding period, abruptly increases at the wandering and decreases to a low level after gut purge. 20-Hydroxyecdysone (20E) increases S6Pase activity in the fat body of feeding larvae, and the activation is dose-dependent. Cell fractionation studies show that S6Pase is mainly associated with the membrane and the optimal pH for membrane-bound S6Pase is 5.5, which is different from that for soluble acid phosphatase (pH 4). Present findings indicate that the S6Pase responsible for a decrease in hemolymph S6P is membrane-bound, and its activity is controlled by a rise of hemolymph ecdysteroid titer at the onset of the wandering stage.     

Inositol trisphosphate mediates the action of hypertrehalosemic hormone on fat body of the American cockroach, Periplaneta americana

The rate of synthesis of inositol trisphosphate (InsP₃) in trophocytes derived from disaggregated cockroach (Periplaneta americana) fat body increases following treatment of the cells with hypertrehalosemic hormone I or II (HTH-I, -II) in vitro. Trophocytes preloaded with [³H]inositol display a significant increase in InsP₃ synthesis as early as 15 s after addition of the hormone. When the trophocytes are pre-incubated with LiCl and subsequently incubated with HTH the [³H] content of the InsP₃ fraction is greater than that found with HTH alone. This is taken as evidence that inositol monophosphate phosphatase is part of the mechanism for clearing InsP₃ from the cytosol. In contrast to HTH, octopamine, which is also capable of exerting a hypertrehalosemic effect in the cockroach, does not increase the synthesis of InsP₃. 1-Octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃), a potent and selective inhibitor of phosphatidylinositol phospholipase C, blocks the activation of phosphorylase by HTH-I as well as the hypertrehalosemic effect induced by the hormone.     

Venom of Euplectrus separatae causes hyperlipidemia by lysis of host fat body cells

Although the lepidopteran larva Pseudaletia separata is attacked by the gregarious ectoparasitoid Euplectrus separatae, it continues to feed and grow. Lipid concentration in the hemolymph of the parasitized host was higher than that of the nonparasitized host from 3 to 8 days after parasitization. Artificial injection of parasitoid venom also elevated lipid concentration in the host hemolymph. One day after venom injection the host's fat body contained many lipid particles, but most of the lipid particles disappeared 7 days later. Light microscopy and transmission electron microscopy showed the lipid particles leaving the fat body cells as a result of the lysis of the fat body cells. These results suggest that the venom elevated the lipid concentration in the host hemolymph by provoking the release of lipid particles from the fat body. Though most of the lipid particles were freely floating in the host hemolymph, a portion of the released lipid particles were phagocytized by hemocytes. The amount of lipid that was loaded to lipophorin in the hemolymph of the venom-injected host was measured, but it was not sufficient to explain the high lipid titer in the hemolymph of parasitized and venom-injected host larvae. The fact that parasitoid larva consumed many hemocytes as evidenced by their presence in the midgut supported the hypothesis that the parasitoid larvae fed on the host hemolymph containing the free lipid particles, the hemocytes phagocytizing the lipid particles, and the lipid-loaded lipophorin. The possibility of the venom contribution to the disruption of the intercellular matrix was examined. The venom showed high activity of matrix metalloproteinase (MMP), especially when it was mixed with the hemolymph of non-parasitized 5th instar larvae. We suggest that the MMP in the venom was activated by some components of the host hemolymph. On the other hand, the venom mixed with hemolymph could not decompose gelatin on zymography, suggesting that the venom-MMP is a different type from gelatinase. Activity of phospholipases A(2), B, C and hyaluronidase were measured with agar plates. High activities of phospholipase B and hyaluronidase were detected. These results suggest that the venom-MMP initially attacked the specific site of the intercellular-matrix of the fat body, and then the hyaluronidase and the phospholipase B cause lysis of the fat body cell, allowing lipid particles to be released into the host hemolymph.     

The pathway of ammonia assimilation in the silkworm,Bombyx mori

Ammonia can easily be assimilated into amino acids and used for silk-protein synthesis in the silkworm, Bombyx mori. To determine the metabolic pathway of ammonia assimilation, silkworm larvae were injected with methionine sulfoximine (MS), a specific inhibitor of glutamine synthetase (GS). Activity of GS in the fat body 2h after treatment with 400&mgr;g MS decreased to less than 10% of the control activity, whereas MS had no effect on the activity of glutamate dehydrogenase (GDH), another enzyme which could possibly be responsible for ammonia assimilation. Glutamine concentration in the hemolymph rapidly decreased after MS treatment, while the ammonia level in the hemolymph sharply increased. Glutamine concentration in the hemolymph 4h after injection decreased with increasing doses of MS, whereas ammonia concentration increased in proportion to the MS dose. MS strongly blocked the incorporation of (15)N label into silk-protein in larvae injected with (15)N ammonia acetate, while it slightly inhibited the incorporation of (15)N-amide glutamine into silk-protein. These results suggest that ammonia is mainly assimilated into glutamine via the action of GS and then converted into other amino acids for silk-protein synthesis and that GDH does not play a major role in ammonia assimilation in B. mori.     

Effects of female wasp accessory secretions, host fat body, and host hemolymph on protein synthesis and egg viability in Microplitis croceipes (Braconidae)

Effects of female wasp reproductive gland secretions, host fat body and hemolymph, and mechanical constriction of the parasitoid egg on protein synthesis were studied in eggs of Microplitis croceipes (Braconidae) dissected from the wasp ovary. Protein synthesis was measured by 35S-methionine incorporation in eggs held in tissue culture medium for 16 h after treatment. Synthesis was stimulated in oocytes obtained from three regions of the ovary (egg tube, reservoir, and calyx) by fat body and venom gland but not by calyx fluid. A combination of fat body, venom gland, and calyx fluid did not enhance the level of synthesis relative to that of fat body or venom gland alone. Host hemolymph inhibited protein synthesis when incubated directly with the dissected eggs but not when the eggs were collected from an artificial oviposition substrate (AOS) containing hemolymph. The inhibitory effect of the hemolymph is thought to be due to the occurrence of melanization. Mechanical constriction did not alter the rate of synthesis, confirming an earlier report that synthesis in newly deposited eggs in ongoing and is not dependent on mechanical activation during the act of oviposition. Mechanisms responsible for sustaining protein synthesis in eggs for 16 h in vitro after their exposure to host hemolymph in the AOSs or fat body and venom gland are not known. Only a small percentage (less than 2%) of dissected ovarial reservoir oocytes that were mechanically constricted and exposed to the venom gland, calyx fluid, and host fat body hatched in vitro. In contrast, an earlier study demonstrated that 38% of eggs oviposited by female wasps into AOSs developed and hatched.     

Control of phospholipase A(2) activity in cockroach (Periplaneta americana) fat body trophocytes by hypertrehalosemic hormone: the role of calcium

Recently, synthetic HTH-I and HTH-II have been shown to increase the formation of free fatty acids in cockroach (Periplaneta americana) fat body. In this study we show that HTH-II increases PLA(2) activity in dispersed trophocytes, thus implying that phospholipid is a potential source of the fatty acids. The increase in HTH-induced PLA(2) activity is triggered by an increase in [Ca(2+)](i) but extracellular Ca(2+) is also required for a maximal Ca(2+) signal: an effect that can be blocked by the introduction of BAPTA into the trophocytes. Treating trophocytes with ryanodine blocks the increase in PLA(2) activity that follows treatment of the cells with HTH-II. This indicates that the Ca(2+) release channels are distinct from those that respond to inositol trisphosphate. Thapsigargin, which releases Ca(2+) to the cytosol from an intracellular store, increases PLA(2) activity. The data show that the enzyme is translocated from the cytosol to the plasma membrane.     

Site of synthesis,tissue distribution,and lipophorin transport of hydrocarbons in Blattella germanica (L.) nymphs

The site of hydrocarbon (HC) synthesis and the amount of HC in various tissues were investigated in relation to developmental stage in the last larval stadium of the German cockroach, Blattella germanica. Abdominal integument linearly incorporated [1-(14)C]propionate into HC for at least 6h in vitro, whereas other body parts synthesized little or no HC. The third through sixth abdominal sternites and tergites were the principal sites of synthesis. High rates of HC synthesis resulted in a fivefold increase in internal HC during the last stadium. We examined the distribution of HC in the hemolymph, fat body, and the developing imaginal cuticle. Hemolymph HC titer was relatively constant at approximately 8&mgr;g/&mgr;l. However, as hemolymph volume increased from 5 to 11&mgr;l in the first 4days of the last stadium, HC content increased and then remained stable the remainder of the stadium. Lipophorin, immunoprecipitated with adult lipophorin polyclonal antibodies, was the only HC carrier protein in nymphal hemolymph and its HC profile was identical to that of hemolymph and similar to that of the epicuticle. The concentration and total amount of hemolymph lipophorin increased until 3days before adult eclosion and declined immediately after ecdysis. The HC content of non-biosynthetic integument (legs, pronotum) doubled during formation of the imaginal cuticle, as did the HC content of sternites, which synthesize HC. HC content of fat body, however, increased threefold during the same period, suggesting that the fat body serves as a storage site for HC during cuticle formation. We conclude that in the last stadium HC is synthesized by abdominal oenocytes, loaded onto hemolymph lipophorin, and transported to fat body and both nymphal and imaginal cuticle. Hydrocarbons associate with the imaginal integument several days before eclosion.     

Preface

The vector of Chagas' disease, Rhodnius prolixus, feeds exclusively on blood. The blood meals are slowly digested, and these insects wait some weeks before the next meal. During the life of an insect, energy‐requiring processes such as moulting, adult gonadal and reproductive growth, vitellogenesis, muscular activity, and fasting, lead to increased metabolism. Carbohydrates are a major source of energy and their mobilization is important. We determined the amounts of glycogen, trehalose, and glucose present in the fat body and/or hemolymph of adult males of R. prolixus and recorded the processes of accumulation and mobilization of these carbohydrates. We also tested our hypothesis that these processes are under endocrine control. The amount of glycogen in the fat body progressively increased until the fourth day after feeding (from 9.3±2.2 to 77. 3±7.5 µg/fat body), then declined to values around 36.3±4.9 µg/fat body on the fifteenth day after the blood meal. Glycogen synthesis was eliminated in decapitated insects and head‐transplanted insects synthesized glycogen. The amount of trehalose in the fat body increased until the sixth day after feeding (from 16. 6±1.7 to 40. 6±5.3 nmol/fat body), decreased abruptly, and stabilized between days 7 and 15 at values ranging around 15–19 nmol/fat body. Decapitated insects did not synthesize trehalose after feeding, and this effect was reversed in head‐transplanted insects. The concentration of trehalose in the hemolymph increased after the blood meal until the third day (from 0.07±0.01 to 0.75±0.05 mM) and at the fourth day it decreased until the ninth day (0.21±0.01 mM), when it increased again until the fourteenth day (0.79±0.06 mM) after the blood meal, and then declined again. In decapitated insects, trehalose concentrations did not increase soon after the blood meal and at the third day it was very low, but on the fourteenth day it was close to the control values. The concentration of glucose in the hemolymph of untreated insects remained low and constant (0.18±0.01 mM) during the 15 days after feeding, but in decapitated insects it progressively increased until the fifteenth day (2.00±0.10 mM). We recorded the highest trehalase activity in midgut, which was maximal at the eighth day after feeding (2,830±320 nmol of glucose/organ/h). We infer that in Rhodnius prolixus, the metabolism of glycogen, glucose, and trehalose are controlled by factors from the brain, according to physiological demands at different days after the blood meal. © 2009 Wiley Periodicals, Inc.     

Clathrin‐dependent endocytosis predominantly mediates protein absorption by fat body from the hemolymph in Bombyx mori

During insect larval–pupal metamorphosis, proteins in the hemolymph are absorbed by the fat body for the maintenance of intracellular homeostasis; however, the type of proteins and how these proteins are internalized into the fat body are unclear. In Bombyx mori, the developmental profiles of total proteins in the hemolymph and fat body showed that hemolymph‐decreased protein bands (55–100 kDa) were in accordance with those protein bands that increased in the fat body. Inhibition of clathrin‐dependent endocytosis predominantly blocked the transportation of 55–100 kDa proteins from the hemolymph into the fat body, which was further verified by RNA interference treatment of Bmclathrin. Six hexamerins were shown to comprise ～90% of the total identified proteins in both the hemolymph and fat body by mass spectrum (MS) analysis. In addition, hemolymph‐specific proteins were mainly involved in material transportation, while fat body‐specific proteins particularly participated in metabolism. In this paper, four hexamerins were found for the first time, and potential proteins absorbed by the fat body from the hemolymph through clathrin‐dependent endocytosis were identified. This study sheds light on the protein absorption mechanism during insect metamorphosis.     

Biosynthesis of apolipophorin-III by the fat body in locusts

Biosynthesis of locust apolipophorin-III (apo-III) was studied in vitro. Gel electrophoresis and immunoblotting analyses of the locust hemolymph demonstrated that apo-III first appears in the hemolymph on the day 3 of the adult stage after the final molt and its hemolymph concentration increases thereafter. When incubated in vitro in a medium containing radioactive amino acid, the fat body cells synthesized the radiolabeled apo-III and released it into the medium. The developmental change in the apo-III synthesizing activity in the fat body reflected that of the apo-III concentration in the hemolymph. RNA isolated from the adult fat body directed the synthesis of apo-III as a major translation product in a cell-free system. These results indicate that the fat body is the tissue responsible for the synthesis of the locust apo-III, and biosynthesis of apo-III is developmentally regulated at the level of mRNA in accordance with the flight activity of the locust.     

丽蝇蛹集金小蜂寄生对寄主蛹可溶性蛋白与芳基蛋白组成与含量的影响

为了探讨蛹期寄生蜂对寄主蛋白代谢的寄生生理效应,利用Bradford蛋白含量测定法、Western免疫印迹法及酶联免疫吸附检测法研究了棕尾别麻蝇Boettcherisca peregrina蛹被丽蝇蛹集金小蜂Nasonia vitripennis寄生后其脂肪体和血淋巴中可溶性蛋白及芳基蛋白组成与含量的变化。结果表明：寄生蛹脂肪体和血淋巴中可溶性蛋白的组成与未寄生相比基本无明显差异; 不论寄生与否寄主蛹脂肪体和血淋巴中芳基蛋白亚基分子量均为80 kDa,该亚基在脂肪体中未出现降解现象,而在血淋巴中仅于寄生后12 h的寄主蛹中呈现2条分子量相近的Western免疫印迹带,说明其降解可能先于未寄生对照。就含量而言,寄生蛹脂肪体中可溶性蛋白含量除寄生后24 h外均显著低于未寄生对照,芳基蛋白含量除寄生后48 h外也均显著低于未寄生对照,其中寄生后12 h的含量仅为未寄生的32.0%。寄生蛹血淋巴中可溶性蛋白含量多低于未寄生蛹,且寄生后2,12,24 h的差异达显著水平;芳基蛋白的含量均有低于未寄生的趋势,其中寄生后12 h的含量为未寄生的17.0%。综合认为,丽蝇蛹集金小蜂的寄生可导致寄主脂肪体和血淋巴中可溶性蛋白及芳基蛋白含量下降。     

In vitro synthesis, release and uptake of storage proteins by the fat body of Manduca sexta: putative hormonal control

1. Two major proteins (P1 and P2) are synthesized by the fifth instar larval fat body of Manduca sexta and then released into the hemolymph. 2. These proteins are later sequestered by the pre-pupal fat body. 20-Hydroxyecdysone does not appear to affect the synthesis of either protein. 3. When day 2 fifth instar larvae are neck-ligated there is an excessive synthesis (supersynthesis) of P2 (arylphorin). 4. Juvenile hormone I (JH I) applications to ligated animals had no effect, but brain homogenate injections resulted in the inhibition of P2 synthesis. 5. Neck ligations of larvae between days 5 and 6 revealed a head critical period between day 5 + 12 hr and day 5 + 18 hr, after which the head is unnecessary for the sequestration of either protein by the fat body. 6. JH I and JH III applications to ligated larvae before the head critical period do not restore the ability of the fat body to sequester the storage proteins. 7. P1 and P2 appear to be synthesized differentially and P2 is sequestered by the fat body to a much lesser extent than P1. 8. P2 is the hemolymph storage protein of both larval and pupal stages, whereas P1 appears to be the storage protein of the pupal fat body. 9. The data indicate that the synthesis of arylphorin and the resorption of both proteins are controlled by a putative head factor(s).     

几种植物源化合物对棉铃虫海藻糖酶活性及相关碳水化合物含量的影响

碳水化合物对昆虫的能量代谢和物质合成具有重要的作用。本研究选用2种一般性生物碱（氢溴酸东莨菪碱和烟碱）以及2种β-葡萄糖苷类化合物（七叶灵和皂角苷）, 研究其在不同浓度下对棉铃虫Helicoverpa armigera (Hübner)幼虫体内海藻糖酶活性及相关碳水化合物代谢的影响。结果表明: 用饲喂法处理3龄幼虫96 h后, 皂角苷对棉铃虫幼虫的活体抑制效果明显, 且随添加物浓度增高, 棉铃虫死亡率上升, 10, 20, 40 g/L浓度下棉铃虫的均重分别是0.194, 0.089和0.034 g, 分别为对照的86.99%, 39.91%和15.24%。对海藻糖酶活性及其相关代谢酶的测定结果表明, 2种苷类化合物显著抑制中肠海藻糖酶活性, 饲喂40 g/L皂角苷的试虫中肠海藻糖酶比活力仅是对照组的54.21%; 饲喂30 g/L七叶灵的试虫中肠海藻糖酶比活力为对照组的83.73%。而2种生物碱类化合物显著抑制血淋巴和脂肪体中海藻糖酶活性, 20 g/L氢溴酸东莨菪碱对棉铃虫血淋巴和脂肪体组织的海藻糖酶活性抑制率分别为7.24%和71.43%; 而20 g/L烟碱对试虫血淋巴和脂肪体组织的海藻糖酶活性抑制率为26.29%和33.44%。用氢溴酸东莨菪碱、 烟碱和七叶灵处理试虫后, 血淋巴海藻糖含量都有所增高。4种化合物能够导致试虫糖原磷酸化酶活性变化, 其中, 皂角苷在中肠和脂肪体表现为显著抑制作用, 而随外源化合物浓度变化, 糖原含量和糖原磷酸化酶活性表现为此消彼长关系。饲喂4种植物源化合物的试虫血淋巴中葡萄糖浓度变化和其海藻糖变化一致。本研究证明β-葡萄糖苷类化合物是海藻糖酶抑制剂, 在作为先导化合物进行农药创制开发方面具有重要意义。     

Synthesis and resorption of a humoral chymotrypsin inhibitor, CI-8, by fat body of the silkworm, Bombyx mori

The Bombyx mori hemolymph contains up to 16 chymotrypsin inhibitors (CIs). The present in vitro culture of tissues in Grace's medium indicated that CI-8, which belongs to the largest molecular-size group of CIs with sugar moiety, is synthesized in the fat body and secreted from it during the feeding period. When the fat body from other strain which synthesizes an allelic component (CI-7) instead of CI-8 was incubated in vitro in hemolymph from the strain which has CI-8, the fat body was found to receive CI-8. Thus it was concluded that CI-8, once secreted into the hemolymph, was again sequestered into the fat body after the onset of spinning. Protein granules isolated from the pupal fat body were shown to contain CI-8, indicating that the sequestered CI-8 is present in the protein granules.